Gap junctions are specialized membrane channels that link the cytoplasms of adjacent cells and facilitate the exchange of hydrophilic ions and small molecules of less than 1kDa in molecular weight between adjacent cells. This intercellular communication results in the functional integration of the cells within a given tissue by the passage of electrical and chemical signals from cell-to-cell. Gap junction channels are formed by a family of proteins, called connexins, which share a common membrane topology. Each connexin is capable of forming gap junction channels with distinct regulatory and conductance properties. Most tissues express more than one connexin and it is postulated that the physiological roles of multiple connexin expression are: (i) to permit the differential expression of distinct connexins in a developmental or tissue-specific manner; (ii) to permit the differential regulation of connexin-specific gap junctional communication by neurotransmitters and hormones acting on the target tissue; (iii) to allow for the differential permeability of gap junction channels to ions and small hydrophilic molecules based on the biophysical properties of the channel pore (e.g. electrical charge, pore diameter); and (iv) to permit the interaction of different cell types at the boundaries of physiologically distinct tissues by the formation of heterologous gap junctions (i.e. two different connexin hemichannels). The proposed studies will examine the development regulation of electrical communication in the embryonic chick heart by autonomic neurotransmitters (alpha-adrenergic, beta-adrenergic, muscarinic cholinergic) and pH. The embryonic chick heart ventricle is known to express three functionally distinct connexins and their expression is developmentally regulated. Double whole cell patch clamp procedures will be performed on cell pairs form primary cultures of 4-, 7-, 14-, and 18-day chick ventricles to provide a quantitative measure of macroscopic and single gap junction channel conductances. The physiological regulation of these developing heart gap junctions will be examined by the superfusion with autonomic receptor agonists and antagonists, membrane-permeable second messenger analogs, protein kinase activators and inhibitors, and pH changes. The differential regulation of the chick connexin42-, connexin43-, and connexin45-specific gap junction channels will be examined using physiologically relevant second messengers. Functional expression of each connexin cDNA is performed by stable transfection in mouse N2A neuroblastoma cells. The N2A cell line is normally devoid of any gap junctions and all three connexins have been successfully expressed in this preparation. The differential permeability of these connexin-specific gap junctions will also be examined using anionic fluorescent dyes of 300 - 500 Da in molecular weight and ion substitutions. As a model for cell-cell interactions expressing distinct connexins, embryonic cardiac myocyte-fibroblast and heterologous connexin N2A cell pairs will be examined using the above procedures. These studies will help determine the physiological roles of multiple connexin expression in intercellular communication with a developing tissue and between neighboring tissues.